Two-stage Strategy for Development of Proteolysis Targeting Chimeras and its Application for Estrogen Receptor Degraders.

Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin-proteasome-system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and linker between them. Generation of efficient PROTACs requires screening of many parameters, especially the length and type of the linkers. We report our proof-of-concept study using a two-stage strategy to facilitate the development of PROTACs against estrogen receptor (ER). In stage-one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ER ligands containing a hydrazide functional group at different positions with a pre-assembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC50= ~10 nM, Dmax= ≥95%). Stage-two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC50=1.1 nM, Dmax=98%) and induced potent anti-proliferation (IC50= 13.2 nM, Imax= 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.